PKN binds and phosphorylates human papillomavirus E6 oncoprotein

The high risk human papillomaviruses (HPVs) are associated with carcinomas of cervix and other genital tumors. Previous studies have identified two vi ral oncoproteins E6 and E7, which are expressed in the majority of HPV-asso ciated carcinomas. The ability of high risk HPV E6 protein to immortalize h uman mammary epithelial cells has provided a single gene model to study the mechanisms of EG-induced oncogenic transformation. In recent years, it has become clear that in addition to EG-induced degradation of p53 tumor suppr essor protein, other targets of E6 are required for mammary epithelial cell s immortalization, Using the yeast two-hybrid system, we have identified a novel interaction of HPV16 E6 with protein kinase PKN, a fatty acid- and Rh o small G protein-activated serine/threonine kinase with a catalytic domain highly homologous to protein kinase C. We demonstrate direct binding of hi gh risk HPV E6 proteins to PKN in wheat-germ lysate in vitro and in 293T ce lls in vivo. Importantly, E6 proteins of high risk HPVs but not low risk HP Vs were able to bind PKN, Furthermore, all the immortalization-competent an d many immortalization-non-competent E6 mutants bind PKN. These data sugges t that binding to PKN may be required but not sufficient for immortalizing normal mammary epithelial cells, Finally, we show that PKN phosphorylates E 6, demonstrating for the first time that HPV E6 is a phosphoprotein, Our fi nding suggests a novel link between HPV E6 mediated oncogenesis and regulat ion of a well known phosphorylation cascade.